ABSTRACT Advancing our understanding of the mechanisms that control bone mass and osteoblast differentiation is crucial to unveil the pathogenesis of skeletal diseases and identify therapeutical approaches for their treatment. Osteoblastic cells operate in a low oxygen (hypoxic) environment. The transcription factors Hypoxia Inducible Factor-1a (HIF1) and HIF2 are critical mediators of the cellular response to hypoxia. Both transcription factors are expressed in cells of the osteoblast lineage. HIF1 was reported to be a positive regulator of bone formation and osteoblast differentiation. Conversely, the role of HIF2 in the control of bone mass and osteoblast biology is still poorly understood. We recently generated mutant mice carrying loss-of-function and gain-of-function mutations of HIF2 in mesenchymal progenitors of the limb bud by using PRX1-Cre. Preliminary analysis of these mutant mice suggested that HIF2 is a negative regulator of osteoblastogenesis and bone formation through a direct action on cells on the osteoblast lineage. Mechanistically, we gathered preliminary evidence that Sox9, which is emerging as a negative regulator of osteoblast differentiation, is likely to mediate the HIF2-dependent impairment of osteoblastogenesis. Our findings constitute a paradigm shift as activation of the HIF signaling pathway has been associated with increased, rather than decreased, osteoblast activity. Moreover, they imply that, as in other cell types, HIF1 and HIF2 have opposing functions in osteoblastic cells. Also, our preliminary data showed that loss of HIF2 in mesenchymal progenitors of the limb bud increases bone mass in both trabecular and cortical bone. HIF2 can be selectively inhibited by small molecules, some of which are currently in clinical trials in patients carrying pathologies associated with high levels of HIF2 activity such as clear cell renal carcinoma. Therefore, determining whether and how HIF2 controls osteoblastogenesis and bone mass not only will expand and deepen our understanding of the role of the hypoxia signaling pathway in the skeleton but could also provide a novel target for the treatment of low bone mass seen in chronic diseases, osteoporosis and with aging. In this proposal, we thus seek to demonstrate that osteoblastic HIF2 regulates bone mass during skeletal development and in adulthood (Aim 1). Moreover, we will establish whether osteoblastic HIF2 controls osteoblastogenesis through a direct action on mesenchymal progenitors and in a Sox9-dependent manner (Aim 2). !